1. Field of the Invention
The present invention relates to a method for determination of hydroxyacetophenone derivatives. More particularly, the present invention relates to a method for determination of hydroxyacetophenone derivatives which comprises measuring hydroxyacetophenone derivatives represented by general formula (I): ##STR1## wherein R represents --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3); and X.sub.1 and X.sub.2 independently represents a halogen atom or hydrogen atom, in association with protein. The present invention also relates to a method for determination of a hydrolase activity utilizing such a measurement method. Hydroxyacetophenone derivatives are useful as the chromogen of synthetic substrates for determining a hydrolase activity. Therefore, by applying the methods described above, the activity of hydrolases such as acid phosphatase or the like can be determined with high sensitivity.
2. Prior Art Statement
In general, hydroxyacetophenone derivatives can be determined by measuring their absorbance 340 nm with a spectrophotometer since the maximum absorption of their absorption spectra exists at about 340 nm. Hydroxyacetophenone derivatives have a structure containing a hydroxy group therein. By utilizing the hydroxy moiety for ester bond or ether bond, the derivatives are expected to be promising as the chromogen of synthetic substrates for determining the hydrolase activity, which are used for clinical inspections.
In recent clinical inspections, the main trend is by means of an automated analyzer. For this reason, synthetic substrates available to analysis of an initial velocity are desired also in activity measurement of enzyme adoptable to automated analysis. With respect to hydrolases such as acid phosphatase, N-acetylglucosaminidase, .beta.-galactosidase, .alpha.-amylase, etc. which have the optimum pH in the range of neutral to acidic regions, however, there are known few chromogens that can be colorimetrically determined in the rang of neutral to acidic regions so that the initial velocity method has not spread very widely. Methods for determination of the activity of these enzymes using conventional synthetic substrates are roughly classified into the following 3 types, in which chromogens used are also illustratively given.
(a) Method which comprises using p-nitrohenylphosphoric acid as substrate, adding an alkali to the chromogenic p-nitrophenol hydrolyzed and released upon enzyme reaction and colorimetrically determining the chromogen quantitatively [Hudson, P. B.: J. Urol., 58, 89 (1947)]
(b) Method which comprises using phenylphosphoric acid as substrate, condensing the similarly released chromogenic phenol with other compounds and colorimetrically determining the chromogen quantitatively [Kind, P. R. N., King, E. J.: J. Clin. Path., 7, 322 (1954)]
(c) Method which comprises using 2,6-dichloro-4-nitrophenylphosphoric acid as substrate and quantitatively determining the chromogenic 2,6-dichloro-4-nitrophenol colorimetrically as it is [Teshima, S., Hayashi, Y., Ando, M.: Clin. Chim. Acta, 168, 231 (1987)]
However, in the methods (a) and (b) described above, a color-forming reaction for termination and condensation are required, respectively so that it is impossible to conduct a rate assay. In the method (c), it is possible to perform the initial velocity analysis. However, substrates obtained using currently found chromogens involve problems that they tend to cause spontaneous hydrolysis and are unstable and furthermore, 2,6-dichloro-4-nitrophenol as the chromogen is measured at about 400 nm and the substrates are thus liable to be affected by the coexisting substances such as bilirubin or hemoglobin in body fluids such as serum or urine.
In this regard, the hydroxyacetophenone derivatives can be expected to as the chromogen of substrates for determination of hydrolases described above, since their wavelength for measurement can be set at about 340 nm which is affected only with difficulty by the coexisting substances.
However, use of the hydroxyacetophenone derivatives alone does not result in sufficiently large absorption at 340 nm. In addition, dissociation of the hydroxy group is also insufficient in the acidic region. Thus, the hydroxyacetophenone derivatives encounter problems that their sensitivity for measurement is poor and the initial velocity method does not satisfactorily work in the acidic region. Such problems remain unsolved yet.
In order to solve the foregoing problems, the present inventors have made extensive investigations and have reached the present invention. That is, the present inventors have made investigations of the use hydroxyacetophenone derivatives represented by general formula (I) described below, in association with protein, in the UV measurement method at about 340 nm. As a result, they have found that there is an increase in the absorption peak at about 340 nm, i.e., an increase in molecular extinction coefficient .epsilon. and acceleration of dissociation of the hydroxy group at the acidic region, i.e., a shift of pKa showing a pH value in 50% dissociation of the hydroxy group in the hydroxyacetophenone derivatives toward the acidic region. They have also found that in this case, the measurement sensitivity can be increased, it becomes possible to perform the measurement at the acidic region, and even in the case of using as the chromogen of substrates for determination of hydrolase activity, the measurement sensitivity can be increased, it becomes possible to perform the initial velocity method at the acidic region.